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| Inhibition of angiogenesis in high glucose environment by calcium dobesilate based on zebrafish model and its mechanism study |
| TANG Zhi-yu DENG Jie-qiang▲ WANG Meng ZUO Ling WU Jin-xia |
| Department of Pulmonary Diseases,Yongchuan Hospital of Traditional Chinese Medicine Affiliated to Chongqing Medical University,Chongqing 402160,China |
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Abstract Objective To observe the inhibitory effect of calcium dobesilate (CaD) on zebrafish embryos angiogenesis and its mechanism.Methods A total of 180 zebrafish embryos were selected and randomly divided into the blank group,the high glucose group (130 mmol/L glucose),the positive drug+high glucose group,the high glucose+CaD(30 μg/mL) group,the high glucose+CaD (60 μg/mL) group,and the high glucose+CaD (90 μg/mL) group.Among them,the high glucose concentration was 130 mmol/L,and the positive drug was 2 mL endothelial cell growth factor receptor tyrosine kinase inhibitor (VRI) at a concentration of 500 ng/mL.CaD in 2 mL was provided in each CaD group at different concentrations.Three doses of 1/10 maximum non-lethal dose (MNLD,30 μg/mL),1/5 MNLD (60 μg/mL),and 3/10 MNLD (90 μg/mL) were selected as the toxicity indicators with reference to lethality experiment.Drug exposure solution in 2 mL was given for continuous 72 h,which was changed every 24 h.The zebrafish embryonic intersegment blood vessel indexes and the number of fluorescence points were observed and calculated under stereoscopic microscope.Fluorescence quantitative polymerase chain reaction was used to detect the expression of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) in embryos after intervention.The contents of VEGF and NOS were determined by enzyme-linked immuno sorbent assay (ELISA).Results The zebrafish embryonic intersegment blood vessel index,the number of microscopic fluorescence points and the expression levels of VEGF and NOS mRNA in the high glucose group were higher than those in the blank group,and the differences were statistically significant (P＜0.05).The zebrafish embryonic intersegment blood vessel indexes and the number of fluorescence points in the positive drug+high glucose group,the high glucose+CaD (60 μg/mL) group,and the high glucose+CaD (90 μg/mL) group were significantly lower than those in the high glucose group (P＜0.01).The CAD administration groups were compared,the blood vessel indexes of zebrafish embryonic intersegment decreased when the CaD administration dose gradually increased,and the differences were significant (P＜0.05).Compared with the high glucose group,the number of fluorescene points of zebrafish embryos in each group showed a downward trend after drug treatment,and the difference was statistically significant (P＜0.05).The mRNA expression levels of VEGF and NOS in the positive drug+high glucose group,the high glucose+CaD (60 μg/mL)group,and the high glucose+CaD (90 μg/mL) group were lower than those in the high glucose group (P＜0.05).There were no significant differences in the VEGF and NOS mRNA expression between the high glucose+CAD (30 μg/mL)group and high glucose group (P＞0.05).The expression levels of VEGF and NOS in the positive drug+high glucose group,the high glucose+CaD (60 μg/mL) group,and the high glucose+CaD (90 μg/mL) group were lower than those in the high glucose group (P＜0.05).There was no significant difference in the expression of NOS and VEGF between the high glucose+CAD (30 μg/mL) group and high glucose group (P＞0.05).Conclusion CaD can inhibit the angiogenesis of zebrafish embryos in high glucose environment,which is related to the inhibition of VEGF/iNOS.
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